1. Field of the Invention
The invention relates to the successful cloning of a full length cDNA of porcine liver ribonuclease inhibitor (RI) gene and its expression in a cellular host. In addition, the invention relates to the cloning of full length rat liver RI, and its expression in a cellular host.
2. Related Art
RI is a cytoplasmic protein ubiquitously present in a variety of mammalian tissues. It inhibits a variety of ribonucleases (RNases) by binding tightly to both intracellular and extracellular RNases by forming a 1: I complex (Roth, J. S., Methods Cancer Res. 3:153-243 (1967); Blackburn, P. et al., J. Biol. Chem. 252:5904-5910 (1977); Blackburn, P., J. Biol. Chem. 254:12484-12487 (1979); Blackburn, P and Moore, S., Enzymes, 3rd Ed. (1982), vol. 15, pp. 317-433; Lee, F. S. et al., Biochemistry 27:8545-8553 (1988)). Some evidence indicates that tissues which are highly active in protein synthesis have a high excess of RI over RNase. Conversely, in catabolically active tissues that do not accumulate RNA, the ratio of RI to RNase is low (Kraft, N. and Shortman, K., Biochim. Biophys. Acta. 217:164-175 (1970)). The biological function of RI has been implicated to be in (a) regulation of RNA turnover by controlling cytoplasmic RNase activity, (b) safeguarding against non-cytoplasmic RNases that mislocalized to the cytoplasm, and (c) regulation of angiogenin, a protein that induces blood vessel growth and contains RNase activity. In vitro, RI is useful in a variety of molecular biology applications where RNase contamination is a potential problem. Examples of these applications include reverse transcription of mRNA, cell-free translation systems, preparation of RNase-free antibodies, and in vitro virus replication. Ideally, RI to be used in these kinds of applications will be capable of inhibiting a large number of RNases, such as eukaryotic RNase A, RNase B and RNase C, as well as prokaryotic RNases.
RI has been purified to homogeneity from several tissues, including placenta (Blackburn, P. et al., J. Biol. Chem. 252:5904-5910 (1977); Blackburn, P., J. Biol. Chem. 254:12484-12487 (1979)), brain (Burton, L. E. et al., Int. J. Pept. Protein Res. 16:359-364 (1980)), and liver (Burton, L. E. and Fucci, N. P., Int. J. Pept. Protein Res. 18:372-379 (1982); Hofsteenge, J. et al., Biochemistry 27:8537-8544 (1988)). The protein has an apparent molecular mass of approximately 50 kilodaltons (kD). The primary amino acid sequence of various RIs, such as human placental (Lee, F. S. et al., Biochemistry 27:8545-8553 (1988)), porcine liver (Hofsteenge, J. et al., Biochemistry 27:8537-8544 (1988)) and rat lung (Kawanomoto, M. et al., Biochim. Biophys. Acta. 1129:335-338 (1992)) is known. The crystal structure of porcine RI has been published (Kobe, B. and Deisenhofer, J., Nature 366:751-756 (1993)). The human placental RI has been successfully cloned and expressed in E. coli (Lee, F. S. et al., Biochemistry 27:8545-8553 (1988); Promega Catalog 1993/1994). A complete rat lung RI cDNA has been described by Kawanomoto et al. (Biochim. Biophys. Acta. 1129:335-338 (1992)); this cDNA has been used to study the distribution of the mRNA in various tissues.
The complete coding sequence of rat lung RI is known, but recombinant rat lung RI has not been expressed in either E. coli or any other known expression host (Kawanomoto, M., et al., Biochim. Biophys. Acta 1129:335-338 (1992)). Several attempts to isolate a cDNA clone for rat liver RI have been unsuccessful (Id).
The complete amino acid sequence of porcine RI has been determined by direct sequencing of the purified protein. In addition, a partial cDNA sequence of porcine kidney RI has been described by Vicentini, A. M. et al. (Biochemistry 29:8827-8834 (1990)). The cDNA lacks 241 nucleotides at the 5'-end of the coding sequence corresponding to the first 81 amino acids of porcine kidney RI. However, a synthetic complete porcine kidney RI coding sequence has been prepared by ligating a synthetic oligonucleotide encoding amino acid residues 1-81 of porcine RI, the sequence of which is based on the amino acid sequence of porcine liver RI, to a restriction fragment of the incomplete cDNA which corresponds to amino acid residues 82-456 of the abovementioned cDNA. This protein has been expressed in Saccharomyces cerevisiae.
From the foregoing, it will be clear that there is a need in the art for recombinantly produced RNase inhibitors that are active against a broad range of RNases from both eukaryotic and prokaryotic sources.